sarcoides and Ascocoryne cylichnium. Descriptions and comparison

FINN ROLL-HANSEN AND HELGA ROLL-HANSEN

Roll-Hansen, F. & Roll-Hansen, H. 1979. and Ascocoryne cylichnium. Descriptions and comparison. Norw. J. Bot. Vol. 26, pp. 193-206. Oslo. ISSN 0300-1156. Descriptions are given and characters evaluated of apothecia and cultures of Ascocoryne sarcoides (Jacq. ex S. F. Gray) Groves & Wilson and A. cylichnium (L. R. Tul.) Korf. The separation into the two may be justified by the occurrence of typical ascoconidiain/4. cylichnium, by a relatively sharp boundary between ectal and medullary excipulum in^4. sarcoides, and to some extent by other characters. F. Roll-Hansen & H. Roll-Hansen, Norwegian Forest Research Institute, Forest Pathology, P. O. Box 62, 1432 As, Norway.

Ascocoryne species have been isolated by many Former descriptions authors from in living spruce trees both in North America and in Europe. In Norway the Ascocoryne sarcoides (Jacq. ex S. F. Ascocoryne species seem to be more common Gray) Groves & Wilson than any other group of fungi in unwounded Groves & Wilson (1967) gave the diagnosis of the stems oiPicea abies. They do not seem to cause Ascocoryne and transferred Octospora any rot or discoloration of importance, but they sarcoides Jacq. ex S. F. Gray ( sarcoides may influence the development of other fungi in [Jacq. ex S. F. Gray] Tul.) to that genus. The the stems. conidial state is Coryne dubia Pers. ex S. F. Great confusion exists regarding identification Gray (Pirobasidium sarcoides Hohn.). Descrip­ of isolates of the Ascocoryne species. In a pre­ tions of the apothecia with the asci and the liminary paper (Roll-Hansen & Roll-Hansen have been given by, for example, 1976) the authors reported isolation of Knapp (1924), Dennis (1956), Gremmen (1960), Ascocoryne sarcoides (Jacq. ex S. F. Gray) Christiansen (1962), and Jahn (1967). The apo­ Groves & Wilson and A. cylichnium (L. R. Tul.) thecia are very similar to those of^4. cylichnium. Korf from stems of . Having found al- What Thind & Singh (1969) described as Coryne lantoid conidia in A. sarcoides - cylichnium (Tul.) Boud. is apparently A. cultures and ovoid conidia in A. cylichnium sarcoides. ascospore-cultures the authors based the Apothecia and conidia-producing stromata in identification of the cultures from the spruce nature. - Knapp (1924) reported that the apo­ stems on these criteria. But later we found both thecia are 8-13 mm wide. Dennis (1956) stated ovoid and allantoid conidia in varying propor­ that the apothecia are 2-10 mm across, the tions in some of the cultures. We therefore paraphyses unbranched, about 1 ^.m thick, realized that a closer comparison between apo­ either enlarged to 2 /xm or abruptly swollen to 4 thecia of A. sarcoides and A. cylichnium, and of fim at the tip, and that the size of the asci is ascospore-cultures from the same ascomata was 90-120 x 8-10 yxm. necessary. The ascospores have been described as el­ In the present paper we first present a short lipsoid, 1-4-celled, and considerably smaller review of some literature data relevant to dis­ than in A. cylichnium. According to Knapp tinguishing between the two species. Afterwards (1924) they are 12-18x4-5 ^m; besides the our own material is treated, and a comparison normal ascospores Knapp also found egg-shaped based on literature and our own data is given. conidia in the asci. Dennis (1956) gave the fol- lowing description: 'Ascospores uniseriate or of the apothecia were given, for example, by biseriate, elliptical or inequilateral, at first non- Knapp (1924), Dennis (1956), Gremmen (1960), septate with 2 oil drops, ultimately 1-septate, Christiansen (1962), and Jahn (1967). occasionally with 3 septa, 10—15(—19) x 3-5 p, Apothecia and indication of conidia-produc- sometimes germinating in situ by a terminal ing stromata in nature. - Knapp (1924) reported germ-tube'. Excipulum of 'C. cylichnium' in his that the apothecia are 4-25 mm wide and that the Fig. 151 fits A. sarcoides better than it fits A. ascospores are mostly 25-30 x 6-7 ton, spore cylichnium. minimum being 20 x 5 tim. In the conidial state, Coryne dubia Dennis (1956) gave the following description: (Pirobasidium sarcoides), some millimetres 'Apothecia like those of C. sarcoides but often high, club-like, or often rather irregular, conidial larger, up to 15 mm. across. Flesh of slender stromata are formed. They have the same colour hyaline hyphae, about lti thick, loosely interwo­ and consistence as the ascomata. The conidia ven in a colourless gelatinous matrix. Excipulum are usually ca. 3.5-6x1 ttm (Hohnel 1902; 40-50 fi thick, of more or less isodiametric, Knapp 1924; Dennis 1956; Christiansen 1962). thin-walled, angular cells, about 8-10 p diame­ Tulasne & Tulasne (1865) stated that ovate (3.5- ter, forming a compact parenchymatous tissue 6.5 x 3 /im) or sometimes rather globose conidia with an irregular outer surface. Asci cylindric- were also formed on the conidial stromata. clavate, long-stalked, conical-truncate at the Hohnel (1902) found, however, that these 'con­ apex, the pore blue in Melzer's reagent, 8- idia' were partly pear-shaped 'basidia' producing spored, about 140-200 x 10-12 p. Ascospores the true conidia (4 x 1 pm), partly roundish cel- biseriate fusiform or inequilateral, equally lulae from which the 'basidia' had grown out. pointed at each end, ultimately multiseptate and Cultures on malt agar. - Gremmen (1960) often abstricting spherical secondary spores, ab­ reported globular 'microconidia' in cultures from out 2 p. across, while still in the ; (14)—18— ascospores of A. sarcoides. He thus apparently 30 x 4-6 p. Paraphyses slender, filiform, slightly obtained a type of conidia which is otherwise enlarged towards the apex, embedded in colour­ supposed to be characteristic of A. cylichnium. less gelatine. On fallen logs and stumps (Fig. Berthet (1964) found rod-shaped, slightly bent 151).' In the figure he drew ectal excipulum and conidia, ca. 5 x 1.5 tim, on the surface in an agar part of medullary excipulum, paraphyses, ascus, culture, but ovoid or subspherical conidia, 2- ascospores, and spherical secondary spores; the 4x2 fim in the agar. Delatour (1976) reported a border between ectal and medullary excipulum similar dimorphism, but he did not mention ('excipulum' and 'flesh') is very sharp, and the whether the roundish conidia were formed in the ascospores are acute at both ends. agar substrate. According to the diagnosis given by Tulasne Etheridge (1957) found no reaction zone on the ascospores are 22-26 x 5-6 tim and rounded gallic acid, except a very intense dark brown at both ends. zone in one case. On tannic acid he found no In most descriptions no conidial state (except reaction zone, or in some isolates light to dark ascoconidia) has been mentioned. But Christ­ brown zones. iansen (1962) reported finds of apothecia of both Coryne cylichnium (Ascocoryne cylichnium) and Coryne sarcoides (Ascocoryne sarcoides) ac­ companied by Pirobasidium sarcoides Hohnel (Coryne dubia Pers. ex S. F. Gray). Usually C. Ascocoryne cylichnium (L. R. Tul.) Korf dubia is accepted to be the conidial state of A. Peziza cylichnium was described by Tulasne sarcoides only. (1853) and transferred to Coryne by Boudier in Cultures on malt agar. - Gremmen (1960) 1907. The combination Ascocoryne cylichnium shortly described cultures of A. cylichnium on was validated by Korf (1971). It is interesting malt agar: 'Culture-work (No. 273): Cultures that Karsten (1885) did not consider it a separate obtained from ascospores develop alike as species but named it Coryne sarcoides *urnalis Coryne sarcoides. In older cultures characteris­ (Nyl.) Karst. (Sarcodea sarcoides *urnalis (Nyl.) tic club-shaped excrescences up to 2.5 mm, Karst. in Not. Sallsk. Faun. Flor. fenn. 11: 232, producing 4 x 1.5 p colourless 1-celled, more or 1870; sarcoides *urnalis (Nyl.) less curved conidia, and reddish coloured micro- Karst. in Myc. Fenn. 1: 87, 1871). Descriptions conidialpustules, were observed.' Gremmen ap- parently happened to get a conidial state cor­ Phenoloxidase production was tested on gallic responding to C. dubia in his cultures from and tannic acid malt agar (Nobles 1948). Growth ascospores of A. cylichnium. rate of the isolates was measured on malt agar in petri dishes in darkness at the respective temp­ eratures. Margin of the mycelium (hypha ends) was marked on four diameters after a few days and again 8 to 10 days later. Conidia from Some Norwegian collections. cultures were measured in lactophenol-cotton­ Descriptions of apothecia and blue under oil immersion (Objective Leitz 90 x, cultures n.a. 1.4). Methods Ascocoryne sarcoides Fresh apothecia from collections of A. sarcoides and A. cylichnium were placed into agar slant Material examined. - Four collections of apo­ tubes or petri dishes to throw their ascospores thecia, thrown-out ascospores, and ascospore- upwards on sterile agar surfaces, and into moist cultures from them were investigated chambers to throw them on clean, flamed slides. thoroughly: one collection from Malus sp., two The ascospores on the agar surfaces provided from Picea abies, and one from Quercus sp. the pure cultures. The ascospores on the slides Microtome sections were made from one of the were mounted in lactophenol-cottonblue for collections from P. abies. In addition one microscoping. In most cases 50 or 20 ascospores ascospore-culture from apothecia on Alnus sp. were measured. Some of the apothecia were and one from apothecia on Be tula sp. were fixed in 70% FA A, embedded in paraffin wax, investigated thoroughly, but in these two cases microtomed 6 to 12 /xm thick in ribbons, stained material was lacking for further descriptions of in safranin-fastgreen and mounted in canada- the apothecia. balsam (Johansen 1940). In addition fruitbodies Apothecia from one collection on Alnus, three from all collections were dried. Hand sections on Be tula, and one on Prunus were also were made from the dry apothecia after moisten­ thoroughly studied, but no cultures had been ing by wrapping them into wet filter paper. taken. Apothecia from 15 other Norwegian col­ Sections from all collections were mounted in lections from nine different hosts were sectioned Melzer's reagent. If by this method the reaction and investigated more quickly. to iodine was negative, then reaction after re­ Apothecia. - In fresh material the diameter of hydration in 5% aqueous KOH solution was the apothecia varied from 2 to 23 mm, the examined (Kohn & Korf 1975; Nannfeldt 1976). consistency was gelatinous, and the colour red­ Some sections were mounted in lactophenol- dish purple (Fig. 1). cottonblue. Ectal excipulum often consisted of two dis­ Also herbarium material was examined from tinct sharply separated layers, but the outer which no cultures had been taken. The asco­ layer was sometimes poorly developed or lack­ spores from these specimens were measured in ing. hand sections of apothecia mounted in lacto­ Outer layer of ectal excipulum was usually of phenol-cottonblue. textura intricata, mostly 0-30 yum thick, consist­ The cultures described were polyascospore- ing of 1-4 fjum thick hyphae in a gel (Figs. 2, 3); it isolates or sometimes (specially indicated) easily weared off. Instead of the gelatinous layer monoascospore-isolates. Culture medium was there was sometimes found a loose layer of short malt agar (1.25% Difco Bacto-Malt Extract + hyphae ending in thin conidiophores forming 2% Difco Bacto-Agar). The cultures were ex­ conidia typical of Coryne dubia, ca. 4-5 x 1 /am amined in different light and temperature condi­ (Fig. 4). tions: Inner layer of ectal excipulum was a 30-100 1. At 15 C and a 12 hour per day photoperiod jam thick pseudoparenchyma, composed of (cool-white fluorescent light + near-UV swollen, roundish, or somewhat angular, light). isodiametric or nearly isodiametric cells, ca. 5-40 2. Constant temperatures in darkness. (50) x 4-20 ,u.m (Figs. 2-7). The cells were small 3. Varying room temperatures at different day­ near the edge of the apothecium, larger and more light in the laboratory. specific farther from the edge (Figs. 6 and 7). It Figs. 1-4. Ascocoryne sarcoides, apothecia. Fig. 1. On Malus sp. x3. Fig. 2. Median section, hymenium with asci and capitate paraphyses, hypothecium, medullary excipulum, ectal excipulum. Hand section in lactophenol-cottonblue x250. Fig. 3. Median section, medullary excipulum, inner and outer layer of ectal excipulum. Microtomed 6 fan x250. Fig. 4. Median section, outer layer of ectal excipulum with conidiophores. Hand section in Melzer's reagent x 500. Figs. 5-8. Ascocoryne sarcoides. Fig. 5. Apothecium, median section of medullary and ectal excipulum. Hand section in Melzer's reagent x 250. Fig. 6. Apothecium, median section. Microtomed 12 ^im x 250. Fig. 7. The same apothecium some distance from the edge, medullary excipulum, ectal excipulum's inner layer, and some rests of the outer layer. Microtomed 12 urn x 250. Fig. 8. Capitate and monilioid paraphyses. Squash slide in Melzer's reagent x500. was characteristic that the inner layer of ectal roundish conidia, 2.0-3.0 x (0.8)1.2-1.6 /am, and excipulum was rather sharply delimitated against all intermediates were often found. We did not the medullary excipulum. find any difference of importance between the Medullary excipulum of textura intricata. conidia formed by air mycelium and those Hyphae 1.5-5(8) /xm thick in a gel (Figs. 2-7). formed submersed in the agar medium. The The paraphyses were unbranched or conidia swelled before they germinated, becom­ branched, 1.5(2) /am thick. The tip was slightly ing broader, ellipsoid, usually measuring 4- to strongly swollen, in some collections only 7x2.5-5.0 jam. 2.0-2.5 /xm, in others capitate and to 6.7 /xm in The growth rate of ascospore-isolates from diameter (Figs. 2, 8). Paraphyses with strongly three collections was 0.7-1.2 mm per 24 hours on swollen tips were sometimes monilioid (Fig. 8). malt agar at 15 C. The asci were 85-130 x 6.5-10 ,xm (Fig. 9), in On gallic acid malt agar reaction was lacking Melzer's reagent with blueing tips after rehydra­ and the radial growth was 1-3 mm in seven days. tion in 5% aqueous KOH solution, but not On tannic acid malt agar the reaction was weak always after rehydration in pure water. to moderately strong (++ to + + +) and the Normal ascospores were 10.1-23.9x3.4-6.1 radial growth was 1-5 mm in seven days. /xm (Fig, 9). The ascospores from seven separate collections varied as shown in Table II as to mean of length and width, and as to minimum Ascocoryne cylichnium and maximum of length and width. The ascospores were 0-3-septate, sometimes 0-1- Material examined. - Sixteen collections of septate. The ends were mostly rounded, but in apothecia (three from Be tula sp. and thirteen some collections more acute (Fig. 10). Abnor­ from Picea abies), thrown-out ascospores, and mal, poorly developed ascospores were also ascospore-cultures from them were investigated found (Fig. 11). In one collection from Quercus thoroughly. Microtome sections were made from sp. there were in addition to normal ascospores seven of the collections from P. abies. Apo­ also shorter, elliptic ones with all transitions to thecia from 39 other Norwegian collections from spherical spores, 5-7 /un in diameter. Normal nine different hosts were hand-sectioned and in­ and abnormal spores occurred together in one vestigated more quickly. ascus. Ascospore-culture was obtained also Apothecia. - In fresh material the diameter of from this specimen. the apothecia varied from 1.3 to 12 mm; they Chains of small, roundish or elongated cells were gelatinous and reddish-purple (Fig. 15), like have been found in some asci (Figs. 11, 12). The those of A. sarcoides. As indicated by the epi­ chains may have been formed by germination of thets 'cylichnium' and 'urnalis' there might be a ascospores, but the appearance often indicated tendency that young apothecia were more cup- that they were primary - a kind of abnormal, shaped in A. cylichnium (Figs. 16, 17) than mA. degenerated ascospores, kept together by re­ sarcoides, where the disc was usually rather flat mains of cytoplasma. The small, roundish cells (Fig. 1). did not separate easily. Ectal excipulum consisted in typical cases of Cultures. - Cultures were compared on malt an outer and an inner layer sharply separated agar. The colour was first white, later usually from each other. lighter or darker violet or greyish-violet. Outer layer of ectal excipulum was of textura The hyphae were septate, 1-6 /am in diameter. intricata, mostly 0-30 /xm thick, consisting of 1^1 Coils of hyphae were sometimes formed. In- /xm thick hyphae in a gel. It was not always crusted hyphae were seen in some cultures. In found. It easily weared off (Fig. 18). Conidio­ all cultures conidiophores occurred on free hy­ phores and conidia were produced from the outer phae in the mycelial mat. Sometimes also coni- layer of an apothecium in a polyascospore- dial stromata were formed (Fig. 13). culture on malt agar; in this case the conidia were In the mycelial mat the conidiophores were both short, roundish, 2-3 x 1.3-1.8 /xm, and al­ usually branched, often Penicillium-like and lantoid, 3.5^1.5x1.0-1.5 /am, with transitions sometimes very complex (Fig. 14). The conidia between the two types. were hyaline and one-celled of varying shape, Inner layer of ectal excipulum (Figs. 18, 19) usually allantoid or rod-shaped, 3.0- was usually 30-100 /xm thick, composed of more 6.0(7.5) x0.8-1.4(1.9) /am; but shorter, more or less swollen, isodiametric or more elongated Figs. 9-13. Ascocoryne sarcoides. Fig. 9. Ascus and ascospores. Hand section in 50% lactic acid x500. Fig. 10. Ascospores thrown out from an apothecium x 1,000. Fig. 11. Asci with normal and abnormal ascospores and chains of small, roundish cells. Squash slide in Melzer's reagent x 1,000. Fig. 12. Ascus with normal ascospores and chains of smaller cells. Squash slide in Melzer's reagent X 1,000. Fig. 13. Conidial stromata in culture on malt agar x6. Figs. 14-19. Fig. 14. Ascocoryne sarcoides, conidiophores and conidia from culture on malt agar. Mounted in lactophenol-cot- tonblue X 1,000. Figs. 15-19. Ascocoryne cylichnium. Figs. 15-16. Two different collections of very small apothecia on Picea abies. Both gave ovoid to allantoid conidia in culture x 6. Fig. 17. Apothecium formed in culture on malt agar. Ascospore-isolate from the same collection as in Fig. 16 x6. Fig. 18. Median section, rather swollen cells in the inner layer of ectal excipulum. Transition zone to medullary excipulum relatively narrow. The ascospores gave cultures with both ovoid and allantoid or rod-shaped conidia (cf. Fig. 28). Microtomed 12 fimx 250. Fig. 19. Median section 2.8 mm from the edge. Transition zone to medullary excipulum broad and indistinct. The ascospores gave cultures with only ovoid conidia (cf. Fig. 27). Microtomed 6/xm X250. cells; there was a fairly even transition to the dark violet or greyish-violet in most cultures. medullary excipulum. The cells near the edge of Two isolates formed ripe apothecia on malt agar. the apothecium were smaller than those farther The hyphae were septate, 1-5 /xm in diameter. from the edge (Figs. 18, 19). They were all Coils of hyphae were often found. transitions from apothecia with rather swollen The conidiophores seldom grew out from hy­ cells in the inner layer of ectal excipulum and phae coils. They were sometimes simple, un- with a rather narrow transition zone (Fig. 18) to branched, but usually branched, often Penicil­ apothecia where swelling was weak and the lium-like (Figs. 26-28). Very complex systems of transition zone broad and indistinct (Fig. 19). conidiophores might be formed. The last-mentioned extreme was typical of strains The conidia were hyaline and one-celled, ex­ of the characterized in culture by broadly tremely varying in shape. But in several cases egg-shaped conidia of even size and shape (Fig. only short, ovoid conidia, often with one big 'oil 27). drop', were found (Figs. 26, 27); the size of these Medullary excipulum of textura intricata. conidia was (2.0)2.3-4.0(5.0) x (1.3)1.5-2.8 /am; Hyphae 1.5-5(8) /am in a gel. but for the separate isolates the variation might The paraphyses were 1.0-2.0 /am thick. The be much less - the conidia might be homogeneous tip was sometimes unswollen but usually more as to shape and size. Young isolates from or less swollen, maximum diameter in different ascospores had in most cases only rather collections varying from 1.5 to 3.0 /am, rarely up homogeneous, short, roundish conidia. to 4.0 /am. The tip was, however, not capitate. In many cases, however, relatively long and The asci were 105-150x 7-10 fxm, always narrow, allantoid or rod-shaped conidia, 3.5- Melzer +. 7.0x0.8-1.2(1.5) /xm, were formed in addition to Normal ascospores were 12-31 x 3.2-7.4 /am. the shorter ones (Fig. 28); sometimes only such The ascospores from the 16 separate collections allantoid or rod-shaped conidia were found. Two varied as shown in Table II regarding mean of examples are given below. From each of three length and width, and minimum and maximum of fruitbodies from separate collections 10 length and width. The ascospores were usually monoascospore-isolates were made. In 29 of pointed at each end and 0, 3, 5(7)-septate (Figs. these isolates only the ordinary ovoid conidia 20, 21). Collections with ascospores having were found. But in one isolate the form and size rounded ends were also found (Fig. 22). In of the conidia were very variable, from broad, several collections none of the spores had more 3-5x2.0-2.6 /am, with one, sometimes two big than three septa. 'oil drops', to narrow ones, 5-6x1.0-1.2 /xm In three collections quite abnormal ascospores (Fig. 29). The last-mentioned isolate had a pure (Fig. 23) were found besides normal-looking white, slow-growing mycelium with irregular ones. Some of them were short, often with contour, in contrast to the other, faster growing, rounded ends and some were spherical, 6-7.5 more or less violet monoascospore-isolates. In /am in diameter. Somewhat angular spores were another case a polyascospore-isolate was made also found. Ascospores from two of the three from an apothecium with 0-7-septate ascospores collections did not germinate. 17.1-24.9 x 4.2-5.8 /xm, often germinating in asci Small conidia or conidia-like cells in asci were by roundish ascoconidia or by hyphae; but in always found in collections of ripe apothecia of culture on malt agar only narrow, allantoid con­ A. cylichnium (Figs. 24, 25). These 'ascoconidia' idia 4x1 /am, were found; ascospores in apo­ were often abundant and conspicuous, but thecia formed on malt agar by this isolate were sometimes scarce and rather difficult to find. 19-23 x 5-5.7/xm. The 'ascoconidia' were undoubtedly often Cultures with the most homogeneous, broad, formed by abstriction from the ascospores, as egg-shaped conidia (Fig. 27) were isolated from depicted by Dennis (1956), but they might some­ apothecia with the broadest and most indistinct times be primary, formed as indicated for A. transition from ectal to medullary excipulum sarcoides. The 'ascoconidia' in A. cylichnium (Fig. 19). There seemed to be a correlation were, however, of even shape and size (ca. between shape of conidia in culture and anatomy 2-2.5 x 1.5-2.0 /am); in squash mounts many of excipulum. were seen as single conidia free from other cells. The growth rate on malt agar for ascospore- Cultures. - Cultures were compared on malt isolates from 11 collections was 0.5-1.7 mm per agar. The colour was first white, later light to 24 hours at 15 C. 0

Figs. 20-25. Ascocoryne cylichnium. Figs. 20-23. Ascospores thrown out from apothecia x 1,000. Fig. 20 Normal Fig 21 H™? IT M18,' ^ RelatiVdy br°ad With r°Unded CndS- Kg- 23- Abnormal- Fi«- 24- Asci with ascofporesSoconidia' Hand^secon m Melzer's reagent x500. - Fig. 25. Ascus with ascospores and ascoconidia. Squash sliSe in Melzer's reagent Figs. 26-29. Ascocoryne cylichnium. Conidiophores and conidia from malt agar cultures. Mounted in lactophenol-cottonblue. Fig. 26. Ovoid conidia x 500. Fig. 27. Ovoid conidia in culture from the apothecium in Fig. 19 x 1,000. Fig. 28. Ovoid and allantoid conidia in culture from the apothecium in Fig. 18 x 1,000. Fig. 29. Monoascospore-isolate. Ovoid conidia, but allantoid conidia from one conidiophore x 1,000. On gallic acid malt agar the reaction was somewhat angular, isodiametric or nearly lacking and the radial growth was 0-3 mm in isodiametric cells with abrupt transition to seven days. On tannic acid malt agar the reaction medullary excipulum. In A. cylichnium the inner was also lacking in all cases except one where it layer of ectal excipulum had more or less of was weak (++); growth rate on this medium was swollen cells; some of them might be 0-2 mm in seven days. isodiametric, but at the same time there were also more elongated cells, and the transition to medullary excipulum was even. There were transitions within A. sarcoides from the extreme Comparison of Ascocoryne sarcoides-type to more cylichnium-like types, sarcoides with Ascocoryne and within A. cylichnium from types recalling A. cylichnium based on literature and sarcoides to types with no isodiametric cells. own data It must be mentioned that our conception of the anatomy of the inner layer of ectal excipulum In the present investigation the anatomy of ex­ contrasts sharply with the figure of ectal and cipulum and the character of the ascoconidia or medullary excipulum in A. cylichnium given by conidia-Iike cells in asci have been considered Dennis (1956, Fig. 151); in our opinion Dennis the two most reliable criteria discriminating be­ depicted a typical A. sarcoides, but he did not tween A. cylichnium and A. sarcoides. The discuss this character. collections have been divided by these criteria Occasionally the outer layer of ectal ex­ into the two species, which have then been cipulum formed conidiophores abstricting al­ described as to other characters elsewhere lantoid conidia (Fig. 4). This was apparently a considered valuable for separation of the two rather reliable specific character for A. species. sarcoides, but it was rarely found. In the following we have compared the dis­ Paraphyses. - Paraphyses which were criminating value of the different characters, strongly swollen and capitate at the tip were using both earlier descriptions and our own found only in A. sarcoides, and paraphyses not observations. at all swollen at the tip only in A. cylichnium. Apothecia where none of the paraphyses were capitate, but more or less swollen at the tips Apothecia were, however, common in both species. There­ Size and shape of the apothecia. - Some au­ fore only the extremes, capitate or completely thors, for example Knapp (1924) and Dennis unswollen paraphyses, had diagnostic value for (1956), found that A. cylichnium produced wider separating the two species. apothecia than A. sarcoides did. In our collec­ Asci. - Generally the asci seemed to be bigger tion it was quite the reverse (Table I). It must be in A. cylichnium than in A. sarcoides (Tab. I). In concluded that width of the apothecia has no many cases size of asci was of little value for discriminating value. The shape of the apothecia separating the species. may, however, on an average be more cupulate Ascospores. - The ascospores have been re­ in A. cylichnium than in A. sarcoides. ported to be smaller in A. sarcoides than in A. Anatomy of excipulum. - Anatomy of ex­ cylichnium, e.g. by Knapp (1924) and Dennis cipulum and ascoconidia were the two (1956). We found less difference than reported characters which we used for separating our own by them (Tab. I). In some collections studied by material into the two species. The diagnostic us the size of the spores was intermediary and of value of these two characters is evident from the no use for separating the two species (Tab. II). fact that we always got the same result indepen­ Generally A. sarcoides has been reported to dent of which of the two characters we used for have ascospores with rounded ends and A. identification. Further, there was a correlation cylichnium spores with acute ends. But the between these two characters on the one side, shape of the spores was very variable. In some and less reliable ones, such as shape of the collections of A. sarcoides many of the spores paraphyses, size of ascospores, and shape of the had rather acute ends, and in some collections of conidia in culture on the other. A. cylichnium the spores had rounded ends. In A. sarcoides was characterized by a distinct both species apothecia with both normal-looking inner layer of ectal excipulum with roundish or and abnormal ascospores were found, often Table I. Size of apothecia, asci, and ascospores given by various authors

sarcoides A. oyliahnium

Diameter of apothecia up to (mm) :

KNAPP (1924) 13 25 DENNIS (1956) 10 15 Our measures 23 12 Asci (um):

DENNIS (1956) 90-120 x 8-10 140-200 x 10-12 Our measures 85-130 x 6.5-10 105-150 x 7-12 Ascospores (um) :

KNAPP (1924) 12-18 x 4-5 23-30 x 6-7 DENNIS (1956) 10-15(19) x 3-5 (14)18-30 x 4-6 Our measures 10-24 x 3.4-6.1 12-31 x 3.2-7.4

within one ascus. It was evident that in many sarcoides as the best character separating it from cases size and form of the ascospores were of no A. cylichnium. In A. cylichnium they have al­ use for separating the two species. ways been found in ripe apothecia, at least in a Ascoconidia. - Ascoconidia have been said to few of the asci (cf. p. 201). The absence or be lacking in A. sarcoides, and to be charac­ presence of such ascoconidia was well corre­ teristic of A. cylichnium. Knapp (1924), however, lated with other, less reliable characters such as reported egg-shaped conidia also in the asci of A. shape of the paraphyses, size of the ascospores, sarcoides. We found chains of small roundish or and shape of the conidia in culture. elongated cells in the asci in two of the collec­ tions of A. sarcoides. But these small cells were Cultures not so even as to size and form as the characteristic ascoconidia found in A. cylich­ No significant difference was found between nium, and they did not easily separate. In fact, cultures of A. sarcoides and A. cylichnium as to besides the anatomy of excipulum, we regard colour and growth rate on malt agar or as to lack of the characteristic ascoconidia in A. appearance and size of the hyphae.

Table II. Variation in size of ascospores among separate collections

Ascospores A., sarcoides A. oyliahnium um via

Total in all collections 10.1-23.9 x 3.4-6.1 12.0-31.0 x 3.2-7.4 Mean in the separate collections 12.8-19.9 x 4.2-5.6 18.7-25.8 x 4.0-6.4 Minimum in the separate collections 10.1-15.8 x 3.4-5.1 12.0-24.0 * 3.2-5.7 Maximum in the separate collections 14.0-23.9 x 4.5-6.1 20.1-31.0 x 5.0-7.4 Table III. Size of conidia in culture on malt agar at ca. 20 C

Shape of conidia H um um

Allantoid or rod-shaped 3.0-6.0(7.5) x 0.8-1.4(1.9) 3.5-7.0 x 0.8-1.2(1.5)

Ovoid 2.0-3.0 x (0.8)1.2-1.6 (2.0)2.3-4.0(5.0) x (1.3)1.5-2.8

Allantoic! or rod-shaped conidia, 3.0- Gremmen, J. 1960. A contribution to the mycoflora of the pine 6.0(7.5) x 0.8-1.4(1.9) /xm, were characteristic of forests in the Netherlands. Nova Hedwigia 1, 251-288 + Tabs. A. sarcoides, but also shorter, more roundish Groves, J. W. & Wilson, D. E. 1967. The nomenclatural status conidia 2.0-3.0 x (0.3)1.2-1.6 /xm were found. of Coryne. Taxon 16, 35^11. Sometimes A. cylichnium formed allantoid or Hohnel, F. von 1902. Fragmente zur Mykologie. I. Mitteilung. rod-shaped conidia similar to those of A. Sber. Akad. Wiss. Wien, Mathem.-Naturw. Classe, Abth. I, 111, 987-1056. sarcoides; occasionally only such conidia were Jahn, H. 1967. pura und Coryne cylichnium in found, but more often they occurred together Westfalen. Westf. Pilzbriefe 6, 162-165. with shorter, more roundish conidia (Tab. Ill, Johansen, D. A. 1940. Plant Microtechnique. McGraw-Hill Figs. 28, 29). Identification of such cultures Book Company, Inc., New York and London 1940. 523 pp. Karsten, P. A. 1885. Revisio monographica atque synopsis seemed often impossible. But only cultures from Ascomycetum in Fennia hucusqve detectorum. Acta Soc. A. cylichnium were found to form exclusively Fauna Flora fenn. 2(6), 1-174. rather uniform, broad, ovoid conidia (Fig. 27). Knapp, A. 1924. Coryne sarcoides (Jacq.) und Var. urnalis Positive reaction on tannic acid malt agar may Nyl. Schw. Zeitschr.f. Pilzkunde 2, 49-53. Kohn, L. M. & Korf, R. P. 1975. Variation in ascomycete indicate A. sarcoides, negative reaction A. iodine reactions: KOH pretreatment explored. Mycotaxon cylichnium. 3, 165-172. Korf, R. P. 1971. Some new discomycete names. Phytologia Acknowledgement. - The authors are grateful to Mr. H. 21(4), 201-207. Gjasrum for critical reading of the manuscript. Nannfeldt, J. A. 1976. Iodine reactions in ascus plugs and their taxonomic significance. Trans. Br. mycol. Soc. 67, 283-287. Nobles, M. K. 1948. Studies in forest pathology. VI. Identifi­ cation of cultures of wood-rotting fungi. Can. J. Res., C, 26, 281^131. References Roll-Hansen, F. & Roll-Hansen, H. 1976. En unders0kelse Berthet, P. 1964. Formes conidiennes de divers Discomycetes. over sopparter som infiserer norsk gran gjennom stam- Bull, trimest. Soc. mycol. Fr. 80, 125-149. mesar, og den soppflora de m0ter i friske traer. En forel0pig Bourchier, R. J. 1964. Laboratory studies on microfungi oversikt. "Rotrota-motet" i Finland, 21.-23. September isolated from the stems of living lodgepole pine, Pinus 1976. 6 pp. + Figs. & Tabs. contorta Dougl. Can. J. Bot. 39, 1373-1385. Thind, K. S. & Singh, H. 1969. The of India - VIII. Christiansen, M. P. 1962. Danish species of the genus Coryne. Proc. Indian Acad. Sci. 70, Sect. B(6), 251-261. Friesia 7(1), 75-85. Tulasne, L.-R. 1853. Nouvelles recherches sur 1'appareil re- Delatour, C. 1976. Microflore interne des tissus ligneux de producteur des champignons. Ann. Sci. Nat., Bot. Ill 20, l'Epicea commun sur pied. I. Inventaire de la microflore 129-182 + PI. naturelle. Ann. Sci. forest. 33, 199-219. Tulasne, L. R. & Tulasne, C. 1865. Selecta fungorum Dennis, R. W. G. 1956. A revision of the British in carpologia 3, 1-206. (Translated to English by W. B. the herbarium of the Royal Botanic Gardens, Kew, with Grove). Paris. notes on related European species. Mycol. Pap. No. 62, 1-216. Received 24 July 1978 Etheridge, D. E. 1957. Comparative studies of Coryne Published September 1979 sarcoides (Jacq.) Tul. and two species of wood-destroying fungi. Can. J. Bot. 35, 595-603.